中文摘要：

目的应用两组三重PCR方法检测非0157：H7型STEC的6种毒力基因stxl、stx2、eae、hly、etpD、katP。方法对多重PCR方法进行改进优化，引入SYBRGreenI荧光定量PCR熔解曲线分析的方法提高工作效率，并结合血清学分析的方法判定结果。建立检测产志贺毒素大肠埃希菌毒力基因的多重PCR方法，简化其分子危害性评估工作，提高检测的准确性与评估效率。结果用模拟样品评估该方法，检测下限分别为：组1stxl基因10ng／ml、stx2基因120ng!ml、eaeP基因110ng／ml；组二etpD基因165ng／ml、katP基因85ng／ml、hly基因15ng／ml。其特异性和灵敏度与单一基因PCR扩增结果基本一致；而运用该方法对210例腹泻患者样品进行检测应用，得到13株非0157STEC菌株，其毒力基因为stxl基因均阳性，eae基因伴随存在，而hly、etpD、katP毒力基因呈散发性分布。结论本研究建立的方法能够快速有效地进行非0157STEC的毒力基因的检出和排查，既提高了检测效率，又可作为评估分子危害性的检查方法。

英文摘要：

Objective Traditional detection approaches for non-O157 STEC are both time and labour consuming in diseases surveillance. Virulence genes detection based on multiplex PCR could not only improve the detection efficiency but also increase the accuracy. Methods Six virulence genes of non- O157：H7（stxl, stx2, eae, hly, etpD, katP6）were detected by two groups of trebling PCRs. The multiplex PCRs were optimized by melting curve analysis in SYBR Green I real-time PCR. Testing result of multiplex PCR was consistent with serological testing. Results The sensitivity limits of the multiplex PCR for stxl, stx2, eaeP, etpD, katP, and hly were 10 ng/ml, 120 ng/ml, 110 ng/ml, 165 ng/ml,85 ng/ml, and 15 ng/ml, respectively, which is similar with that of single PCR. When the multiplex PCR was applied in 120 adults and 90 children diarrhea samples detection, 13 cases were detected for non-O157 positive. Conclusion The method we established can be used for non-O157 STEC virulence genes detection and screening with high efficiency and accuracy.